Medium composition for preparing botulinum toxin

ABSTRACT

The present invention relates to a medium composition for production of botulinum toxin and, more particularly, to a medium composition for culture of  Clostridium  sp. capable of producing botulinum toxin. The medium composition of the present invention comprises a porcine peptone and at least one plant-derived peptone selected from the group consisting of a garden pea hydrolysate, a cotton seed hydrolysate and a wheat gluten hydrolysate. When the medium according to the present invention, which contains plant-derived peptones, porcine peptones and minerals, is used for culture of  Clostridium botulinum,  the growth rate of the bacterium in the medium is higher than that in each of the medium that is in current use and the medium comprising plant-derived peptones alone. In addition, when the medium of the present invention is used, a high concentration of botulinum toxin can be produced by culturing the bacterium in a safe manner

TECHNICAL FIELD

The present invention relates to a medium composition for production ofbotulinum toxin and, more particularly, to a medium composition forculture of strains of Clostridium capable of producing botulinum toxin.The medium composition of the present invention comprises a porcinepeptone and at least one plant-derived peptone selected from the groupconsisting of a garden pea hydrolysate, a cotton seed hydrolysate and awheat gluten hydrolysate.

BACKGROUND ART

A variety of Clostridium strains that secrete neurotoxic toxins havebeen discovered since 1890s, and the characterization of toxins that aresecreted from these bacteria has been made for the past 70 years(Schant, E. J. et al., Microbiol. Rev., 56:80, 1992).

Neurotoxic toxins derived from the Clostridium sp., that is, botulinumtoxins, are classified into seven types (types A to G) depending ontheir serological properties. Each of the toxins has a toxin proteinhaving a size of about 150 kDa and naturally comprises a complex ofseveral non-toxic proteins bound thereto. A medium complex (300 kDa) iscomposed of a toxin protein and a non-toxic non-hemagglutinin protein,and a large complex (450 kDa) and a very large complex (900 kDa) arecomposed of the medium-sized complex bound to hemagglutinin (Sugiyama,H., Microbiol. Rev., 44:419, 1980). Such non-toxic hemagglutininproteins are known to function to protect the toxin from low pH andvarious proteases in the intestines.

The toxin is synthesized as a single polypeptide having a molecularweight of about 150 kDa in cells, and then cleaved at a position of 1/3starting from the N-terminal end by the action of intracellular proteaseor treatment with an artificial enzyme such as trypsin into two units: alight chain (L; molecular weight: 50 kDa) and a heavy chain (H;molecular weight: 100 kDa). The cleaved toxin has greatly increasedtoxicity compared to the single polypeptide. The two units are linked toeach other by a disulfide bond and have different functions. The heavychain binds to a receptor of a target cell (Park. M. K. et al., FEMSMicrobiol. Lett., 72:243, 1990) and functions to interact with abiomembrane at low pH (pH 4) to form a channel (Mantecucco, C. et al.,TIBS., 18:324, 1993), and the light chain has the pharmacologicalactivity of interfering the secretion of neurotransmitters, when it ispermeable to cells by using detergent or introduced to cells byelectroporation or etc (Poulain, B. et al., Proc. Natl. Acad. Sci. USA.,85:4090, 1988).

The toxin inhibits the exocytosis of acetylcholine at the cholinergicpresynapse of a neuromuscular junction to cause asthenia. It has beenconsidered that even treatment with a very small amount of the toxinexhibits toxicity, suggesting that the toxin has any enzymatic activity(Simpson, L. L. et al., Ann. Rev. Pharmacol. Toxicol., 26:427, 1986).

According to a recent report, the toxin has metallopeptidase activity,and its substrates include composed of synaptobrevin, syntaxin, asynaptosomal associated protein of 25 kDa (SNAP25), etc., which are theunit proteins of an exocytosis machinery complex. Each type of toxinuses one of the above-described three proteins as its substrate, and itis known that type B, D, F and G toxins cleave synaptobrevin at aspecific site, type A and E toxins cleave SNAP25 at a specific site, andtype C cleaves syntaxin at a specific site (Binz, T. et al., J. Biol.Chem., 265:9153, 1994).

Particularly, type A botulinum toxin is known to be soluble in a diluteaqueous solution at a pH of 4.0-6.8. It is known that a stable non-toxicprotein is separated from neurotoxin at a pH of about 7 or higher, andas a result, the toxicity is gradually lost. Particularly, it is knownthat the toxicity decreases as pH and temperature increase.

The botulinum toxin is fatal to the human body even in small amounts andis easy to produce in large amounts. Thus, it constitutes four majorbio-terror weapons together with Bacillus anthracis, Yersinia pestis andsmallpox virus. However, it was found that, when type A botulinum toxinis injected at a dose that does not systematically affect the humanbody, it can paralyze local muscle in the injected site. Based on thischaracteristic, type A botulinum toxin can be used in a wide range ofapplications, including winkle removing agents, agents for treatingspastic hemiplegia and cerebral palsy, etc. Thus, the demand for type Abotulinum toxin has increased, and studies on methods of producingbotulinum toxin so as to satisfy the demand have been activelyconducted.

A current typical commercial product is BOTOX® (a purified neurotoxincomplex of type A botulinum toxin) that is commercially available fromAllergan, Inc., USA. A 100-unit vial of BOTOX® is composed of about 5 ngof a purified neurotoxin complex of type A botulinum toxin, 0.5 mg ofhuman serum albumin and 0.9 mg of sodium chloride and is reconstitutedusing sterile saline without a preservative (injection of 0.9% sodiumchloride). Other commercial products include Dysport® (a complex ofClostridium botulinum type A toxin and hemagglutinin, which has lactoseand human serum albumin in a pharmaceutical composition containingbotulinum toxin and is reconstituted using 0.9% sodium chloride beforeuse) that is commercially available from Ipsen Ltd., UK, MyoBloc® (aninjectable solution (a pH of about 5.6) comprising botulinum type Btoxin, human serum albumin, sodium succinate and sodium chloride) thatis commercially available from Solstice Neurosciences, Inc.

A medium for culture of Clostridium botulinum, which is generally usedin a method for production of botulinum toxin as disclosed in KoreanPatent No. 10-1339349, contains animal components. Thus, if an animalabnormal prion known as an agent that causes transmissible spongiformencephalopathy is contained in the animal components due tocontamination, it poses problems in a process for producing botulinumtoxin.

Transmissible spongiform encephalopathy (TSE) is known as aneurodegenerative disorder causing serious degeneration of neurons, andexamples thereof includes bovine spongiform encephalopathy (BSE),Scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinkersyndrome, Kuru, transmissible mink encephalopathy, chronic wastingdisease, feline spongiform encephalopathy, etc., which affect humans andanimals It was reported that BSE crosses the species barrier and infectseven humans.

The agent that causes transmissible spongiform encephalopathy (TSE) hascharacteristics in that it has no immunogenicity and the incubationperiod is long. From histopathological analysis of BSE-affected bovinebrain tissue, it can be seen that special spongiform vacuoles wereformed in the brain due to damage to neurons and deposition of abnormalprotein fibers.

The cause of TSE is a proteinaceous infectious particle known as theabnormal prion. Unlike general viruses that require nucleic acid, theabnormal prion is an infectious particle composed of protein alonewithout comprising nucleic acid. Regarding TSE, it is known that, whenan abnormal prion (PrPsc) that is an infectious particle binds to anormal prion (PrPc), it is converted to a pathogenic prion which is thenaccumulated in the brain (Prusiner S B, Alzheimer Dis Assoc Disord.,3:52-78, 1989).

Creutzfeldt-Jakob disease is a rare neurodegenerative disorder of humantransmissible spongiform encephalopathy (TSE) where the transmissibleagent is apparently an abnormal isoform of a prion protein. Anindividual with Creutzfeldt-Jacob disease can deteriorate from apparentperfect health to akinetic mutism within six months. Thus, a potentialrisk may exist of acquiring a prion mediated disease, such asCreutzfeldt-Jacob disease, from the administration of a pharmaceuticalcomposition which contains a biologic, such as a botulinum toxin,obtained using animal-derived products. Thus, if a pharmaceuticalcomposition is prepared by drug substance produced using animal-derivedcomponents, it can subject the patient to a potential risk of receivingvarious pathogens or infectious agents.

Under this technical background, the present inventors have found that,when a medium comprising transmissible spongiformencephalopathy(TSE)-free plant-derived peptone, mineral and TSE-freeporcine peptone(e.g., TSE-certificated porcine peptone) is used forculture of Clostridium botulinum in order to prevent the risk ofdeveloping the above-described prion-mediated disease, the risk ofdevelopment of the prion-mediated disease that can occur in a mediumthat is in current use (original medium) can be excluded, and the growthrate of Clostridium botulinum in the medium can be increased compared tothat in the medium that is in current use and the medium comprisingplant-derived peptone alone, thereby completing the present invention.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a medium compositioncomprising a plant-derived peptone having no risk of transmissiblespongiform encephalopathy (TSE) infection and a porcine peptone havingno risk of TSE infection, and a method for production of botulinumtoxin, which improves the production of botulinum toxin by culturingClostridium botulinum in the medium composition.

Technical Solution

To achieve the above object, the present invention provides a mediumcomposition for culture of Clostridium botulinum, the medium compositioncomprising: at least one plant-derived peptone selected from the groupconsisting of a garden pea hydrolysate, a cotton seed hydrolysate and awheat gluten hydrolysate; and a porcine peptone.

The present invention also provides a method for producing botulinumtoxin, comprising the steps of: (a) culturing Clostridium botulinumusing the medium composition to produce botulinum toxin; and (b)recovering the produced botulinum toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth of Clostridium botulinum in a medium (APFmedium) comprising plant-derived peptone.

FIG. 2 shows the growth of Clostridium botulinum in a medium comprisingplant-derived peptones, minerals, amino acids and vitamins.

FIG. 3 shows the results of examining whether a precipitate is formedafter sterilization of a medium comprising plant-derived peptones,minerals, amino acids and vitamins is sterilized.

FIG. 4 shows the results of examining whether a precipitate is formedafter sterilization of a medium comprising plant-derived peptones andminerals.

FIG. 5 shows the growth of Clostridium botulinum in media obtained byadditionally adding vitamins, amino acids and “BD Recharge™ withoutGlucose and L-Glutamine” to media for culture of the bacterium, whichcontain plant-derived peptone and mineral.

FIG. 6 shows the growth of Clostridium botulinum in media for culture ofthe bacterium, which contain various kinds of plant-derived peptones.

FIG. 7 shows contour plots of FFD for mineral screening, and responseoptimization. FIG. 7a contour plot for high setting; FIG. 7b contourplot for middle setting; FIG. 7c contour plot for low setting; and FIG.7d response optimization for maximum OD.

FIG. 8 shows contour plots of FFD for mineral screening, and responseoptimization. FIG. 8a contour plot for high setting; FIG. 8b contourplot for middle setting; FIG. 8c contour plot for low setting; and FIG.8d response optimization for maximum OD.

FIG. 9 shows contour plots for plant peptone screening, and responseoptimization. FIG. 9a contour plot for middle setting; FIG. 9b contourplot for low setting; and FIG. 9c response optimization for maximum OD.

FIG. 10 shows the growth curve of Clostridium botulinum in the finallyselected APF medium, and a change in toxin concentration.

FIG. 11a shows a response surface plot and response optimization, andFIG. 11b for screening of the porcine peptone Primatone P37 or Bactoproteose peptone No. 3.

FIG. 12 graphically shows time-dependent OD values comparing the growthof Clostridium botulinum between a medium that is in current use, amedium comprising plant-derived peptones (APF medium) and a final mediumcomprising plant-derived peptones and porcine peptones (APF+porcine).

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, an animal protein-free (APF) mediumcomposition showed an increased growth rate of Clostridium botulinumcompared to a medium that is in current use (original medium), but itwas taken into account to add medium components that further increasethe growth rate of the bacterium and have no risk of infection with TSEor the like. Thus, a porcine peptone (e.g., a TSE-certificated porcinepeptone), which has not been reported to cause TSE infection, was addedto the APF medium, and the growth of a bacterium in the APF medium wasexamined. As a result, the APF medium showed an increased growth rate ofthe bacterium compared to a medium that is in current use and a mediumcomprising plant-derived peptones alone. Thus, if the above medium isused, a high concentration of botulinum toxin can be produced byculturing a bacterium in a safe manner under TSE-free conditions.

It was reported that TSE affects humans and various animals, includinggoats, sheep, minks and deer, but a TSE outbreak in pigs has not yetbeen reported (Jahns H et al., Vet Rec., 159(5):137-142, 2006; Kofler Met al., Schwweiz Arch Tierheild, 148(7):341-342, 344-348, 2006). Thus,in the present invention, porcine peptone was added to a mediumcomposition for culture of Clostridium botulinum, thereby preparing amedium composition showing an increased growth rate of the bacteriumcompared to a medium that is in current use and a medium comprisingplant-derived peptone alone in a process for production of a biologicalagent such as a botulinum toxin without the risk of causing aprion-mediated disease such as Creutzfeldt-Jakob disease.

As used herein, the term “medium that is in current use or originalmedium” means a medium comprising casein hydrolysate, yeast extract andthioglycollate medium, which are animal-derived medium components. Theterm “APF medium (animal protein-free medium)” means a medium thatcontains no animal-derived protein and that contains plant-derivedpeptones, minerals and glucose.

In an example of the present invention, in order to produce botulinumtoxin by culturing Clostridium botulinum under transmissible spongiformencephalopathy (TSE)-free conditions, an APF medium comprising TSE-freeplant-derived peptone was prepared and compared with a medium that is incurrent use (containing an animal component). As a result, it could beseen that an optimal medium composition for culturing Clostridiumbotulinum is one comprising a plant-derived peptone, at least onemineral selected from the group consisting of KH₂PO₄, K₂HPO₄ andNa₂HPO₄, and a carbon source (e.g., glucose, and the optimal growth ofthe bacterium in this medium was found. As a result, as shown in Table13, it was determined that the optimal contents of plant-derivedpeptones in the finally selected medium composition for culture ofClostridium botulinum are 5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cottonand 5 g/L HyPep™ 4601N, and the optimal contents of minerals in themedium composition are 5.5 g/L K₂HPO₄ and 3 g/L Na₂HPO₄.

In another example of the present invention, the growth pattern ofClostridium botulinum in the finally selected APF medium containingplant-derived peptones and minerals and the toxin concentration weremeasured. As a result, as shown in Table 12 and FIG. 10, the OD valuestarted to increase after 12 hours of culture of Clostridium botulinum,and at 24 hours of culture, the culture medium showed an OD_(540 nm) of3.5465 and an OD_(600 nm) of 3.0695. Then, the OD value decreasedgradually, and at 48 hours of culture, the culture medium showed anOD_(540 nm) of 0.792 and an OD_(600 nm) of 0.7224. The toxinconcentration in the culture supernatant of Clostridium botulinumstarted to increase after 5 hours of culture and showed a final value of31.41 μg/ml. When the toxin concentration was measured after rupturingthe bacterium, the toxin started to be produced after 5 hours ofculture, and the toxin concentration continued to increase, wasmaintained at a uniform level after 28 hours of culture, and showed afinal value of 38.39 μg/ml.

In another example of the present invention, the growth of Clostridiumbotulinum in a medium comprising plant-derived peptones and porcinepeptones was examined As a result, as shown in Table 14, when thebacterium was cultured in the medium further comprising porcine peptone,the medium showed an OD value higher than the medium comprisingplant-derived peptone alone. Particularly, the medium showing thehighest growth rate of the bacterium was a medium comprising 10 g/LPrimatone P37 and 10 g/L Bacto proteose peptone No. 3, which showed anOD_(540 nm) value of 4.951.

In another example of the present invention, the growth pattern ofClostridium botulinum in the medium comprising plant-derived peptonesand porcine peptones was examined As a result, as shown in Table 15, at20 hours of culture of the strain, the growth of the bacterium in themedium comprising plant-derived peptones and porcine peptones wasactive, and the medium showed an OD value which was about 11 timeshigher than that of the commercial medium and about 2.2 times higherthan that of the medium comprising plant-derived peptone alone. At 29hours of culture of the bacterium, the peak OD value of the mediumcomprising plant-derived peptones and porcine peptones was at least twotimes higher than that of each of the medium that is in current use andthe medium comprising plant-derived peptones alone. Thus, it could beseen that the growth rate of the bacterium in the medium comprisingplant-derived peptones and porcine peptones was highest among those inthe three types of media.

As a result, as shown in Table 16, it was determined that the optimalcontents of plant-derived peptones in the finally selected mediumcomposition for culture of Clostridium botulinum, which contains atransmissible spongiform encephalopathy (TSE)-free porcine peptone, are5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cotton and 5 g/L HyPep™ 4601N, andthe optimal contents of minerals in the medium composition are 5.5 g/LK₂HPO₄ and 3 g/L Na₂HPO₄, the content of the glucose is 10 g/L, and thecontents of porcine peptone are 13 g/L Primatone P37 and 13 g/L Bactoproteose peptone No. 3.

Therefore, in one aspect, the present invention is directed to a mediumcomposition for culture of Clostridium botulinum, the medium compositioncomprising: at least one plant-derived peptone selected from the groupconsisting of a garden pea hydrolysate, a cotton seed hydrolysate and awheat gluten hydrolysate; and a porcine peptone.

As used herein, the term “plant-derived peptone” means a peptoneextracted from garden pea, cotton seed or wheat gluten. Preferably, theplant-derived peptone may be commercially available Hy-Pea™ 7404,UltraPep™ Cotton, HyPep™ 7504 or HyPep™ 4601N, but is not limitedthereto. The term “porcine peptone” means a component extracted from aporcine tissue. Preferably, the porcine peptone may be a porcine peptonecomprising about 54.91-60.69 wt % of peptides having a molecular weightof 500 Da or less, or a porcine peptone comprising about 38.48-42.53 wt% of peptides having a molecular weight of 500 Da or less. Morepreferably, the casein hydrolysate may be commercially availablePrimatone P37 or Bacto proteose peptone No. 3, but is not limitedthereto.

As used herein, the term “plant-derived peptone” or “plant-derivedhydrolysate” means a product obtained by degrading a protein isolatedfrom a plant. For example, the garden pea peptone (garden peahydrolysate) means a product obtained by degrading a total proteinisolated from garden pea. In addition, the term “porcine peptone” or“porcine hydrolysate” means a product obtained by degrading porcineprotein.

Degradation of the plant-derived protein or the porcine tissue proteinis preferably performed by partial digestion. Degradation of the proteinis preferably performed by acid treatment, base treatment, enzymetreatment, high-pressure treatment, heat treatment or physicaltreatment. More preferably, the porcine peptone may be one obtained byenzyme treatment. The physical treatment is, for example, grinding.

The plant-derived peptone or porcine peptone that is used in the presentinvention is a partial degradation product of protein, is a mixturecomprising not only amino acids that are single molecules, but alsopeptides composed of several to several tens of amino acids, and intactprotein molecules.

In the present invention, the content of the plant-derived peptone inthe medium composition may be 0.1-10 w/v % (1-100 g/L), preferably 0.2-5w/v % (2-50 g/L), more preferably 0.5-2 w/v % (5-20 g/L).

In the present invention, the medium composition contains all the gardenpea hydrolysate, the cotton seed hydrolysate and the wheat glutenhydrolysate, and the content ratio of the garden pea hydrolysate, thecotton seed hydrolysate and the wheat gluten hydrolysate in the mediumcomposition may be 1:0.24-43.62:0.01-50.57 by weight, preferably1:0.68-14.46:0.09-9.87 by weight, more preferably 1:1.6-2.4:0.6-1.4 byweight.

In the present invention, the content of the porcine peptone in themedium composition may be 0.2-10 w/v % (2-100 g/L), preferably 0.4-5 w/v% (4-50 g/L), more preferably 1-2 w/v % (10-20 g/L).

In the present invention, the porcine peptone may be a hydrolysatecomprising about 54.91-60.69 wt % of peptides having a molecular weightof 500 Da or less and/or a hydrolysate comprising about 38.48-42.53 wt %of peptides having a molecular weight of 500 Da or less.

In the present invention, if the porcine peptone comprises both ahydrolysate comprising about 54.91-60.69 wt % of peptides having amolecular weight of 500 Da or less and a hydrolysate comprising about38.48-42.53 wt % of peptides having a molecular weight of 500 Da orless, the content (by weight) of the porcine peptone may be calculatedusing the following equation 1, and the content ratio of the hydrolysatecomprising about 54.91-60.69 wt % of peptides having a molecular weightof 500 Da or less and the hydrolysate comprising about 38.48-42.53 wt %of peptides having a molecular weight of 500 Da or less may preferablybe 1:0.8-1.2 by weight.

B≧−0.625*A+12.5, B≦−1.019*A+53   Equation 1

wherein

A: the content (0-5.2 w/v % (0-52 g/L)) of the hydrolysate comprisingabout 54.91-60.69 wt % of peptides having a molecular weight of 500 Daor less;

B: the content (0-5.3 w/v % (0-53 g/L)) of the hydrolysate comprisingabout 38.48-42.53 wt % of peptides having a molecular weight of 500 Daor less.

In the present invention, the medium composition for culture ofClostridium botulinum may further contain a carbon source and at leastone mineral selected from the group consisting of K₂HPO₄ (dipotassiumphosphate), Na₂HPO₄ (disodium phosphate) and KH₂PO₄ (monopotassiumphosphate).

Herein, examples of the carbon source include, but are not limited to,monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g.,maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g.,dextrin, cyclodextrin, starch, etc.), sugar alcohols (e.g., xylitol,sorbitol, erythritol, etc.).

In the present invention, the content of the mineral in the mediumcomposition may be 0.05-3.5 w/v % (0.5-35 g/L), preferably 0.1-1.75 w/v% (1-17.5 g/L), and more preferably 0.25-0.7 w/v % (2.5-7 g/L).

In another aspect, the present invention is directed to a method forproducing botulinum toxin, comprising the steps of: (a) culturingClostridium botulinum using the above-described medium composition toproduce botulinum toxin; and (b) recovering the produced botulinumtoxin.

In the present invention, the culturing may be performed under anaerobicconditions, and the botulinum toxin may be selected from the groupconsisting of botulinum toxin types A, B, C, D, E, F and G.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are illustrative purposesonly and are not to be construed to limit the scope of the presentinvention. Thus, the substantial scope of the present invention will bedefined by the appended claims and equivalents thereof.

Example 1 Culture of Clostridium botulinum in Plant-Derived PeptoneMedium

1-1: Composition of a Medium Currently Used in Culture

The reagents and medium components used in the present invention werepurchased from Sigma (USA), Kerry Inc. (USA), BD Biosciences (USA),Gibco Life Technologies (USA), and Quest (USA).

A medium that is in current use having a composition comprising 2%casein hydrolysate (20 g/L), 1% yeast extract (10 g/L), 1% glucose (10g/L) and 0.5% thioglycollate medium (5 g/L) was used for the seedculture and main culture of Clostridium botulinum to produce botulinumtoxin. 5 g of the thioglycollate medium per liter of the medium that isin current use is composed of 2.52 g of an enzymatic digest of casein,0.84 g of yeast extract, 0.925 g of dextrose, 0.085 g of sodiumthioglycollate, 0.42 g of NaCl, 0.085 g of L-cysteine, 0.00014 g ofResazurin and 0.125 g of bacteriological agar.

1-2: Composition of APF Medium Used in Culture

A negative control medium was prepared by removing casein hydrolysate,yeast extract and thioglycollate medium from the medium that is incurrent use (original medium) for culture of Clostridium botulinum, andan animal protein-free (APF) medium was prepared by adding fourplant-derived peptone candidates (Hy-Pea™ 7404, UltraPep™ Cotton, HyPep™7504, and HyPep™ 4601N) to the negative control medium (Table 1).

Table 1 shows the components of the plant-derived peptone-comprising APFmedium for culture of Clostridium botulinum in comparison with themedium that is in current use.

TABLE 1 Conc. medium that is Negative APF Components of Medium (g/L) incurrent use Control Medium Glucose 10 10 10 10 Sodium Chloride (NaCl)0.42 0.42 0.42 0.42 Casein hydrolysate 20 20 — — Yeast extract 10 10 — —Thioglycollate medium 5 5 — — Hy-Pea ™ 7404 20 — — 20 UltraPep ™ Cotton10 — — 10 HyPep ™ 7504 10 — — 10 HyPep ™ 4601N 10 — — 10

1-3: Seed Culture of Clostridium botulinum

20 μl of Clostridium botulinum (the Korean Centers for Disease Controland Prevention Accession No.: 4-029-CBB-IS-001) was inoculated into aculture tube containing 10 ml of a sterile medium having each of thecompositions described in Examples 1-1 and 1-2 and was subjected toprimary seed culture (stationary culture) at 35° C. for 22-30 hoursunder anaerobic conditions. When the growth of the bacterium in theprimary seed culture was confirmed, 8 ml of the primary seed culture wasinoculated into a 1-liter culture bottle containing 800 ml of a sterilemedium having the same medium composition and was subjected to secondaryseed culture (stationary culture) at 35° C. for 8-15 hours underanaerobic conditions.

1-4 : Main Culture of Clostridium botulinum

In order to produce a botulinum toxin by culturing Clostridiumbotulinum, the main culture of the bacterium was performed.Specifically, 9.3 L of a medium having each of the compositionsdescribed in Examples 1-1 and 1-2 was prepared and placed in a 10-literincubator, followed by sterilization of the medium. Nitrogen wassupplied to make anaerobic conditions, and the growth of the bacteriumwas performed at a temperature of 35° C. and an agitation speed of 50rpm.

The secondary seed culture in the 1-liter culture bottle in Example 1-3was inoculated into a 10-liter incubator through an inoculation lineconnected to the inoculation port of the 10-liter incubator. Clostridiumbotulinum in the 10-liter incubator was cultured under the conditions of35° C. and 50 rpm and the set culture conditions were monitored andrecorded. When the bacterium was cultured for 100 hours or more, themain culture was terminated.

The growth of Clostridium botulinum in the animal protein-free (APF)medium prepared by adding four plant-derived peptone candidates (Hy-Pea™7404, UltraPep™ Cotton, HyPep™ 7504, and HyPep™ 4601N) to the negativecontrol medium was compared with that of the bacterium in the negativecontrol medium prepared by removing casein hydrolysate, yeast extractand thioglycollate medium from the medium that is in currentuse(original medium) (Table 1).

As a result, as shown in Table 1 and FIG. 1, Clostridium botulinum didnot grow in the negative control medium, but started to grow in theoriginal medium (medium that is in current use) at 24 hours afterinoculation of the bacterium and started to grow in the plant-derivedpeptone-containing medium at 30 hours after inoculation of thebacterium.

Example 2 Culture of Clostridium botulinum in Medium ContainingPlant-Derived Peptone, Mineral, Amino Acid and Vitamin

Because the growth of Clostridium botulinum in the medium prepared byadding four plant-derived peptones in Example 1 was slower than that inthe original medium, solutions thereto were provided as follows.

1) To examine the effect of thioglycollate functioning to make anaerobicconditions, thioglycollate was removed from the original medium (mediumthat is in current use), and a change in the growth rate of thebacterium in the thioglycollate-free medium was analyzed.

2) Because the slower growth rate could be because of the lack of thenitrogen source, the peptone concentration in the medium used forculture of the bacterium was increased two times.

3) The growth of Clostridium botulinum in a medium obtained by addingmineral, amino acid and vitamin to the plant-derived peptone-containingmedium was compared with the growth of Clostridium botulinum in an APFmedium disclosed in U.S. Pat. No. 8,012,716 (Allergan) (Table 2).

Table 2 shows the components of the medium for culture of Clostridiumbotulinum, which contains plant-derived peptones, minerals, amino acidsand vitamins.

TABLE 2 medium 1 2 3 4 APF that is (APF (APF (APF (APF Medium of incurrent Medium Medium Medium Medium Allergan Components of Medium g/Luse Candidate) Candidate) Candidate) Candidate) Company Glucose 10 10 1010 10 10 15 Sodium Chloride (NaCl) 0.42 0.42    0.42    0.42    0.42   0.42 — Casein hydrolysate 20 20 20 — — — — Yeast extract 10 10 10 — —— 12 Thioglycollate medium 5 5 — — — — — Hy-Pea ™ 7404 20 — — 20 40 20 —UltraPep ™ Cotton 10 — — 10 20 10 — HyPep ™ 7504 10 — — 10 20 10 —HyPep ™ 4601N 10 — — 10 20 10 — KH₂PO₄ 7 — — — —  7 — K₂HPO₄ 5.5 — — — —  5.5 — Na₂HPO₄ 5 — — — —  5 — MgSO₄7H₂O 10 — — — — 10 — Vitamin Kit100X — — — — 1X — Amino acid mixture 50X — — — — 1X — Soy peptone 32.5 —— — — —   32.5

As a result, as shown in Table 2 and FIG. 2, when the bacterium wascultured in the medium that is in current use without thioglycollate,the growth rate of the bacterium in the medium was slower than that inthe thioglycollate-containing medium, indicating that thioglycollateinfluences the growth rate of the bacaterium. When the peptoneconcentration in the medium was increased two times, the bacterium didnot grow in the medium. When in the case in which mineral components,amino acids and vitamins were added to the peptone-containing medium,the growth rate of the bacterium was similar to that in the medium thatis in current use, but a precipitate was formed after sterilization ofthe medium. In addition, it was seen that the growth rate of thebacterium in the Allergan's APF medium was similar to that in the mediumthat is in current use.

Example 3 Production of Precipitate by Sterilization of MediumContaining Plant-Derived Peptones, Minerals, Amino Acid and Vitamin

In Example 2, it was observed that the growth rate of Clostridiumbotulinum in the medium containing plant-derived peptones, minerals,amino acids and vitamins, among the APF medium candidates 2 to 4 shownin Table 2, was similar that in the medium that is in current use.However, formation of a precipitate appeared after sterilization of themedium, and thus the cause thereof was examined (Table 3).

Table 3 shows the components of a medium for culture of Clostridiumbotulinum, which was used in sterilization and contains plant-derivedpeptones, minerals, amino acids and vitamins.

TABLE 3 medium 1 2 3 that is (APF (APF (APF 4 in current Medium MediumMedium (APF Medium Components of Medium g/L use Candidate) Candidate)Candidate) Candidate) Glucose 10 10 10 10 10 10 Sodium Chloride (NaCl)0.5 0.5 0.5 0.5   0.5   0.5 Casein hydrolysate 20 20 — — — — Yeastextract 10 10 — — — — Thioglycollate medium 5 5 — — — — Hy-Pea ™ 7404 20— 20 20 20 20 UltraPep ™ Cotton 10 — 10 10 10 10 HyPep ™ 7504 10 — 10 1010 10 HyPep ™ 4601N 10 — 10 10 10 10 KH₂PO₄ 7 — 7 7 — — K₂HPO₄ 5.5 — 5.55.5 — — Na₂HPO₄ 5 — 5 5 — — MgSO₄7H₂O 10 — 10 10 — — Vitamin Kit 100X —1X — 1X 1X (Adding after Sterilization) Amino acid mixture 50 X — 1X —1X 1X (Adding after Sterilization)

As a result, as shown in Table 3 and FIG. 3, only in the case in whichminerals were added to the plant-derived peptone-containing medium, aprecipitate was formed after sterilization of the medium, indicatingthat the main cause of formation of the precipitate was the minerals.This is believed to be because the mineral components interacted withone another under the conditions of high temperature and high pressureduring sterilization of the medium.

Example 4 Formation of Precipitate by Sterilization of Medium ContainingPlant-Derived Peptones and Minerals

In order to identify the mineral components involved in the formation ofprecipitate caused by sterilization as confirmed in Example 3, variouscombinations of different components were added to media, followed bysterilization (Table 4).

Table 4 shows the components of media for culture of Clostridiumbotulinum, which contain plant-derived peptones and minerals, and theresults of sterilization of the media.

TABLE 4 medium that is 1 2 3 4 5 6 in (APF (APF (APF (APF (APF (APFComponents of current Medium Medium Medium Medium Medium Medium Mediumg/L use Candidate) Candidate) Candidate) Candidate) Candidate)Candidate) Glucose 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5   0.5  0.5   0.5   0.5   0.5   0.5 (NaCl) Casein hydrolysate 20 20 — — — — —— Yeast extract 10 10 — — — — — — Thioglycollate 5 5 — — — — — — mediumHy-Pea ™ 7404 20 — 20 20 20 20 20 20 UltraPep ™ Cotton 10 — 10 10 10 1010 10 HyPep ™ 7504 10 — 10 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 1010 10 KH₂PO₄ 7 — —  7 —  7  7  7 K₂HPO₄ 5.5 — —   5.5   5.5 —   5.5  5.5 Na₂HPO₄ 5 — —  5  5  5 —  5 MgSO₄7H₂O 10 — — 10 10 10 10 —precipitation, x ∘ ∘ ∘ ∘ x aggregation 7 8 9 10 11 12 (APF (APF (APF(APF (APF (APF Components of Medium Medium Medium Medium Medium MediumMedium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate)Glucose 10 10 10 10 10 10 Sodium Chloride   0.5   0.5   0.5   0.5   0.5  0.5 (NaCl) Casein hydrolysate — — — — — — Yeast extract — — — — — —Thioglycollate — — — — — — medium Hy-Pea ™ 7404 20 20 20 20 20 20UltraPep ™ Cotton 10 10 10 10 10 10 HyPep ™ 7504 10 10 10 10 10 10HyPep ™ 4601N 10 10 10 10 10 10 KH₂PO₄ —  7  7 — — 10 K₂HPO₄ — —   5.5  5.5   5.5 — Na₂HPO₄  5 — —  5 —  5 MgSO₄7H₂O 10 10 — — 10 —precipitation, ∘ x x x ∘ x aggregation

As a result, as shown in Table 4 and FIG. 4, among the media containingplant-derived peptones and minerals, the medium containing MgSO₄.7H₂Oand K₂HPO₄ and the medium containing MgSO₄.7H₂O and Na₂HPO₄ formed aprecipitate after sterilization.

Example 5 Culture of Clostridium botulinum under Conditions in which noPrecipitate is Formed in APF Medium

An experiment was performed to determine whether culture of Clostridiumbotulinum is possible when vitamin and amino acid are additionally addedto the APF medium of Example 4containing plant-derived peptones andminerals. In addition, an experiment was performed to examine whetherculture of the bacterium is possible in a medium which is free ofplant-derived peptone and mineral and contains vitamins, amino acidsand/or “BD Recharge™ without Glucose and L-Glutamine” (Cat No. 670002,BD Bioscience) (a yeast extract-based medium component free of glucoseand L-glutamine) (Table 5).

Table 5 shows the components of media obtained by additionally addingvitamins, amino acids and “BD Recharge™ without Glucose and L-Glutamine”to the medium for culture of Clostridium botulinum, which containsplant-derived peptones and minerals, and the growth rates of thebacterium in the media.

TABLE 5 medium that is 1 2 3 4 5 6 in (APF (APF (APF (APF (APF (APFComponents of current Medium Medium Medium Medium Medium Medium Mediumg/L use Candidate) Candidate) Candidate) Candidate) Candidate)Candidate) Glucose 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5   0.5  0.5   0.5   0.5   0.5   0.5 (NaCl) Casein 20 20 — — — — — —hydrolysate Yeast extract 10 10 — — — — — — Thioglycollate 5 5 — — — — —— medium Sodium 1 — — — — — — — thioglycollate Hy-Pea ™ 7404 20 — 20 2020 20 20 20 UltraPep ™ 10 — 10 10 10 10 10 10 Cotton HyPep ™ 7504 10 —10 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 10 10 KH₂PO₄ 7 — —  7 7  7 —  7 K₂HPO₄ 5.5 — —   5.5 —   5.5   5.5 — Na₂HPO₄ 5 — —  5 — —  5 5 MgSO₄7H₂O 10 — — — 10 — — — Vitamin Kit 100X — — 1X 1X 1X 1X 1X Aminoacid — — 1X 1X 1X 1X 1X mixture 50 X w/o Glucose and 45.42 — — — — — — —L-glutamine Growth x ∘ x ∘ ∘ ∘ Details Growing Growing Growing Growingin in in in 24 hrs 24 hrs 24 hrs 24 hrs 7 8 9 10 11 12 (APF (APF (APF(APF (APF (APF Components of Medium Medium Medium Medium Medium MediumMedium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate)Glucose 10 10 10 10 10 10 Sodium Chloride   0.5   0.5   0.5   0.5   0.5  0.5 (NaCl) Casein — — — — — — hydrolysate Yeast extract — — — — — —Thioglycollate — — — — — — medium Sodium — —  1 — — — thioglycollateHy-Pea ™ 7404 20 20 20 20 — — UltraPep ™ 10 10 10 10 — — Cotton HyPep ™7504 10 10 10 10 — — HyPep ™ 4601N 10 10 10 10 — — KH₂PO₄ — — — — — —K₂HPO₄ — — — — — — Na₂HPO₄ — — — — — — MgSO₄7H₂O — — — — — — Vitamin Kit100X 1X — — 1X — 1X Amino acid 1X — — 1X — 1X mixture 50 X w/o Glucoseand —   45.42 —   45.42   45.42   45.42 L-glutamine Growth x x x x x ∘Details Growing in 48 hr

As a result, as shown in Table 5 and FIG. 5, only in the case in whichthe medium contained plant-derived peptones and a combination of two ormore minerals of KH₂PO₄, K₂HPO₄ and Na₂HPO₄ and further containedvitamin and amino acid, Clostridium botulinum grew within 24 hours afterinoculation of the bacterium. In addition, in the case in which themedium was free of plant-derived peptone and mineral and containedvitamins, amino acids and “BD Recharge™ without Glucose andL-Glutamine”, the bacterium grew within 48 hours after inoculation ofthe bacterium. In conclusion, the most suitable medium composition forculture of Clostridium botulinum comprises plant-derived peptones,KH₂PO₄, K₂HPO₄, Na₂HPO₄, amino acids and vitamins.

Example 6 Culture of Clostridium botulinum in Media Containing DifferentPlant-Derived Peptones

An experiment was performed to examine whether culture of Clostridiumbotulinum is possible when different combinations of plant-derivedpeptones are added to the APF medium of Example 5.

Table 6 shows the components of media for culture of Clostridiumbotulinum, which contain different plant-derived peptones, and theresults of examining whether the bacterium grew in the media.

TABLE 6 medium that is 1 2 3 4 5 6 in (APF (APF (APF (APF (APF (APFcurrent Medium Medium Medium Medium Medium Medium Components of Mediumg/L use Candidate) Candidate) Candidate) Candidate) Candidate)Candidate) Glucose 10 10 10 10 10 10 10 10 Sodium Chloride (NaCl) 0.50.5   0.5   0.5   0.5   0.5   0.5   0.5 Casein hydrolysate 20 20 — — — —— — Yeast extract 10 10 — — — — — — Thioglycollate medium 5 5 — — — — —— Sodium thioglycollate 0.1 — — — — — — — Hy-Pea ™ 7404 10 — 10 10 — — —— UltraPep ™ Cotton 10 — 10 — 10 — — — HyPep ™ 7504 10 — 10 — — 10 — 10HyPep ™ 4601N 10 — 10 — — — 10 10 KH₂PO₄ 7 —  7  7  7  7  7  7 K₂HPO₄5.5 —   5.5   5.5   5.5   5.5   5.5   5.5 Na₂HPO₄ 5 —  5  5  5  5  5  5MgSO₄7H₂O 10 — — — — — — — Vitamin Kit 100X — — 1X 1X 1X 1X 1X Aminoacid mixture 50 X — — 1X 1X 1X 1X 1X w/o Glucose and 45.42 — — — — — — —L-glutamine Growth ∘ ∘ ∘ ∘ ∘ ∘ 7 8 9 10 11 12 13 (APF (APF (APF (APF(APF (APF (APF Medium Medium Medium Medium Medium Medium MediumComponents of Medium Candidate) Candidate) Candidate) Candidate)Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 SodiumChloride (NaCl)   0.5   0.5   0.5   0.5   0.5   0.5   0.5 Caseinhydrolysate — — — — — — — Yeast extract — — — — — — — Thioglycollatemedium — — — — — — — Sodium thioglycollate — — — — —   0.1 — Hy-Pea ™7404 — — 10 10 10 — — UltraPep ™ Cotton 10 10 — — 10 — — HyPep ™ 7504 —10 — 10 — — — HyPep ™ 4601N 10 — 10 — — — — KH₂PO₄  7  7  7  7  7 —  7K₂HPO₄   5.5   5.5   5.5   5.5   5.5 —   5.5 Na₂HPO₄  5  5  5  5  5 —  5MgSO₄7H₂O — — — — — — — Vitamin Kit 100X 1X 1X 1X 1X 1X 1X 1X Amino acidmixture 50 X 1X 1X 1X 1X 1X 1X 1X w/o Glucose and — — — — — 45.42 45.42L-glutamine Growth ∘ ∘ ∘ ∘ ∘ x x

As a result, as shown in Table 6 and FIG. 6, even when only or two ofthe four plant-derived peptones were added to the medium, culture ofClostridium botulinum was possible.

Taking the results of Examples 5 and 6 into account, it could be seenthat at least one plant-derived peptone should be contained in themedium and that the plant-derived peptone cannot be substituted with “BDRecharge™ without Glucose and L-Glutamine” (Cat No. 670002, BDBioscience) (a yeast extract-based medium component free of glucose andL-glutamine).

Example 7 Experiment for Selection of Two of Three Minerals Contained inMedium

In Examples 1 to 7, it was determined that the APF medium compositionused for culture of Clostridium botulinum comprises glucose, sodiumchloride (NaCl), four plant-derived peptones, three minerals, aminoacids, and vitamins. Among these medium components, medium componentshaving no significant effect on the growth of the bacterium were removedto reduce the number of the medium components. Thus, it was judged thatamino acids and vitamins have no significant effect on the growth ofClostridium botulinum, and under this judgment, amino acids and vitaminswere removed from the medium components. In addition, in order to selecttwo of three minerals, the bacterium was cultured using the mediumcompositions shown in Table 7, and the OD (540 nm and 600 nm) values at24 hours and 48 hours after inoculation of the bacterium were measuredand compared.

Table 7 shows the compositions of media resulting from the first-stageselection of minerals and the growth of Clostridium botulinum in themedia.

TABLE 7 medium that is 1 2 3 4 5 in (APF (APF (APF (APF (APF Componentsof current Medium Medium Medium Medium Medium Medium g/L use Candidate)Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein hydrolysate 2020 — — — — — Yeast extract 10 10 — — — — — Thioglycollate 5 5 — — — — —medium Hy-Pea ™ 7404 10 — 10 10 10 10 10 UltraPep ™ Cotton 10 — 10 10 1010 10 HyPep ™ 7504 10 — 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 10KH₂PO₄ 7 — — 7 — 7 — K₂HPO₄ 5.5 — — — 5.5 5.5 — Na₂HPO₄ 5 — — — — — 5Culture 24 hr OD 540 nm 0.942 −0.017 −0.024 4.396 3.226 4.218 600 nm0.780 −0.016 −0.020 3.832 2.691 3.593 Culture 48 hr OD 540 nm 2.459−0.014 −0.019 4.716 5.220 3.502 600 nm 2.057 −0.015 −0.018 3.852 4.2882.989 6 7 8 9 10 11 (APF (APF (APF (APF (APF (APF Components of MediumMedium Medium Medium Medium Medium Medium Candidate) Candidate)Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein hydrolysate — — —— — — Yeast extract — — — — — — Thioglycollate — — — — — — mediumHy-Pea ™ 7404 10 10 10 10 10 10 UltraPep ™ Cotton 10 10 10 10 10 10HyPep ™ 7504 10 10 10 10 10 10 HyPep ™ 4601N 10 10 10 10 10 10 KH₂PO₄ 7— 7 3.5 3.5 3.5 K₂HPO₄ — 5.5 5.5 2.75 2.75 2.75 Na₂HPO₄ 5 5 5 2.5 2.52.5 Culture 24 hr OD 3.214 4.964 3.991 3.951 3.938 3.594 2.680 4.3043.351 3.341 3.335 3.036 Culture 48 hr OD 5.460 2.056 2.603 5.726 5.6825.434 4.480 1.587 2.020 4.688 4.647 4.459

As a result, as shown in Table 7, at 24 hours of inoculation of thebacterium, the medium that is in current use showed an OD(540 nm) valueof 0.942, and the APF medium containing K₂HPO₄ and Na₂HPO₄ showed thehighest OD(540 nm) value of 4.964 among the APF media. In addition, at48 hours after inoculation of the bacterium, the APF medium containingKH₂PO₄ and Na₂HPO₄ showed the highest OD value and active bacterialgrowth.

Meanwhile, as shown in FIG. 7, contour plots of K₂HPO₄ and Na₂HPO₄having high main effects were drawn. As a result, as the concentrationsof K₂HPO₄ and Na₂HPO₄ increased, the OD value increased. And Clostridiumbotulinum showed the highest growth when minerals were added to themedium at the concentrations of KH₂PO₄=0 g/L, K₂HPO₄=5.5 g/L, andNa₂HPO₄=5 g/L.

Meanwhile, in order to confirm the results of bacterial cultureaccording to more precise addition of minerals, a second-stageexperiment was performed using a response surface methodology. Becausethe medium composition cannot have a negative value, the experiment wasplanned using a CCF (central composite faced) design and performed byculturing the bacterium in the medium compositions shown in Table 8.Then, the experimental results were combined with the results of thepreviously performed FFD and subjected to statistical analysis.

Table 8 shows the compositions of media obtained by the second-stageselection of minerals and the growth of Clostridium botulinum in themedia.

TABLE 8 medium that 1 2 3 4 5 6 7 8 9 is (APF (APF (APF (APF (APF (APF(APF (APF (APF in Components of Medium Medium Medium Medium MediumMedium Medium Medium Medium current Medium g/L Candidate) Candidate)Candidate) Candidate) Candidate) Candidate) Candidate) Candidate)Candidate) use Glucose 10 10 10 10 10 10 10 10 10 10 10 Sodium Chloride0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 — (NaCl) Casein hydrolysate 20 —— — — — — — — — 20 Yeast extract 10 — — — — — — — — — 10 Thioglycollatemedium 5 — — — — — — — — — 5 Hy-Pea ™ 7404 10 10 10 10 10 10 10 10 10 10— UltraPep ™ Cotton 10 10 10 10 10 10 10 10 10 10 — HyPep ™ 7504 10 1010 10 10 10 10 10 10 10 — HyPep ™ 4601N 10 10 10 10 10 10 10 10 10 10 —KH₂PO₄ 7 — 7 3.5 3.5 3.5 3.5 3.5 3.5 3.5 — K₂HPO₄ 5.5 2.75 2.75 — 5.52.75 2.75 2.75 2.75 2.75 — Na₂HPO₄ 5 2.5 2.5 2.5 2.5 — 5 2.5 2.5 2.5 —OD 24 hr 540 nm 4.408 3.587 2.233 4.639 1.778 4.332 3.904 3.907 4.0461.556 600 nm 3.836 3.086 1.896 4.068 1.503 3.777 3.366 3.368 3.505 1.307OD 48 hr 540 nm 5.021 5.760 4.359 3.594 4.529 4.054 6.492 5.621 5.4733.622 600 nm 4.284 4.925 3.695 3.049 3.832 3.457 5.603 4.830 4.677 3.062

Contour plots were drawn and used for comparison. As shown in FIG. 8,the OD value increased as the concentration of KH₂PO₄ decreased. Whenthe optimal conditions were compared, the results were different fromthe results of FFD due to the curvature effect, and the value of K₂HPO₄was the same, but the value of Na₂HPO₄ changed from 5 g/L to 3.1313 g/L.Thus, it was confirmed that the optimal mineral conditions of the mediumby statistical analysis are 5.5 g/L K₂HPO₄ and 3 g/L Na₂HPO₄.

Example 8 Experiment for Selection of Plant-Derived Peptones Containedin Medium

As shown in Tables 9 and 10, plant-derived peptones were combinedaccording to a mixture design, and the growth of Clostridium botulinumin a medium containing the combined plant-derived peptones was examined

Table 9 shows the compositions of media obtained by the first-stageselection of plant-derived peptones and the growth of Clostridiumbotulinum using the media.

TABLE 9 4 5 6 7 8 9 10 11 medium 1 2 3 (APF (APF (APF (APF (APF (APF(APF (APF that (APF (APF (APF Medium Medium Medium Medium Medium MediumMedium Medium is in Components of Medium Medium Medium Candi- Candi-Candi- Candi- Candi- Candi- Candi- Candi- current Medium g/L Candidate)Candidate) Candidate) date) date) date) date) date) date) date) date)use Glucose 10 10 10 10 10 10 10 10 10 10 10 10 10 Sodium Chloride 0.50.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 — (NaCl) Casein 20 — — — — —— — — — — — 20 hydrolysate Yeast extract 10 — — — — — — — — — — — 10Thioglycollate 5 — — — — — — — — — — — 5 medium Hy-Pea ™ 7404 10 5 10 55 — — 6.667 6.667 — — 10 — UltraPep ™ 10 5 — 5 5 10 — 6.667 6.667 20 —10 — Cotton HyPep ™ 7504 10 5 10 5 5 — 10 — 6.667 — 20 10 — HyPep ™4601N 10 5 — 5 5 10 10 6.667 — — — 10 — K₂HPO₄ 5.5 5.5 5.5 5.5 5.5 5.55.5 5.5 5.5 5.5 5.5 5.5 — Na₂HPO₄ 3 3 3 3 3 3 3 3 3 3 3 3 — OD 24 hr 540nm 3.541 2.440 3.345 3.305 3.317 2.852 3.695 2.772 2.353 1.688 4.8422.239 600 nm 3.058 2.066 2.868 2.831 2.853 2.445 3.183 2.376 2.014 1.4194.245 1.893 OD 48 hr 540 nm 0.811 0.935 0.731 0.799 1.400 0.777 1.6601.090 1.810 1.402 2.093 3.341 600 nm 0.714 0.795 0.647 0.694 1.199 0.6801.403 0.929 1.548 1.210 1.764 2.812

Table 10 shows the compositions of media obtained by the second-stageselection of plant-derived peptones and the growth of Clostridiumbotulinum using the media.

TABLE 10 1 2 3 4 5 6 7 (APF (APF (APF (APF (APF (APF (APF Components ofMedium Medium Medium Medium Medium Medium Medium Medium g/L Candidate)Candidate) Candidate) Candidate) Candidate) Candidate) Candidate)Glucose 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.50.5 0.5 (NaCl) Casein 20 — — — — — — — hydrolysate Yeast extract 10 — —— — — — — Thioglycollate 5 — — — — — — — medium Hy-Pea ™ 7404 10 5 5 — —10 10 5 UltraPep ™ 10 5 5 10 6.667 10 — 5 Cotton HyPep ™ 7504 10 5 5 106.667 — — 5 HyPep ™ 4601N 10 5 5 — 6.667 — 10 5 K₂HPO₄ 5.5 5.5 5.5 5.55.5 5.5 5.5 5.5 Na₂HPO₄ 3 3 3 3 3 3 3 3 OD 24 hr 540 nm 3.425 3.6402.349 2.581 3.272 1.289 3.514 600 nm 2.969 3.159 2.029 2.244 1.096 1.0963.032 OD 48 hr 540 nm 0.769 0.836 1.633 0.961 1.501 1.148 0.803 600 nm0.675 0.732 1.420 0.854 1.270 0.982 0.698 medium that 8 9 10 11 12 13 is(APF (APF (APF (APF (APF (APF in Components of Medium Medium MediumMedium Medium Medium current Medium Candidate) Candidate) Candidate)Candidate) Candidate) Candidate) use Glucose 10 10 10 10 10 10 10 SodiumChloride 0.5 0.5 0.5 0.5 0.5 0.5 — (NaCl) Casein — — — — — — 20hydrolysate Yeast extract — — — — — — 10 Thioglycollate — — — — — — 5medium Hy-Pea ™ 7404 20 — 6.667 10 10 10 — UltraPep ™ — — — 10 10 10 —Cotton HyPep ™ 7504 — — 6.667 10 10 10 — HyPep ™ 4601N — 20 6.667 10 1010 — K₂HPO₄ 5.5 5.5 5.5 5.5 5.5 5.5 — Na₂HPO₄ 3 3 3 3 3 3 — OD 24 hr0.776 1.257 3.457 5.376 5.235 4.809 2.208 0.649 1.098 2.950 4.689 4.5344.246 1.863 OD 48 hr 0.880 1.278 0.962 1.986 1.994 2.010 3.185 0.7441.124 0.818 1.708 1.710 1.717 2.708

As a result, as shown n FIG. 9, contour plots were drawn and used foranalysis. It was determined that HyPep™ 7504 corresponding to thecomponent C has the lowest effect on the growth of Clostridiumbotulinum. Based on this determination, HyPep™ 7504 was excluded frommedium components. In conclusion, it was determined that the compositionof the finally selected plant-derived peptones that are contained in themedium comprises 5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cotton and 5 g/LHyPep™ 4601N.

Example 9 Culture of Clostridium botulinum in Medium Containing or NotContaining NaCl

The medium compositions used in Examples 1 to 8 contained a small amount(0.5 g/L) of NaCl. In order to examine the growth of Clostridiumbotulinum according to the concentration change of NaCl, the content ofNaCl in the medium was adjusted to a range from 0 to 1 g/L, followed byculture of the bacterium in the medium.

Table 11 shows the components of NaCl-containing media for culture ofClostridium botulinum and the growth of Clostridium botulinum in themedia.

TABLE 11 1 2 3 4 5 6 7 8 9 (APF (APF (APF (APF (APF (APF (APF (APF (APFComponents of Medium Medium Medium Medium Medium Medium Medium MediumMedium Medium g/L Candidate) Candidate) Candidate) Candidate) Candidate)Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 1010 10 10 Sodium Chloride 0.5 — — — 0.5 0.5 0.5 1 1 1 (NaCl) Hy-Pea ™7404 5 5 5 5 5 5 5 5 5 5 UltraPep ™ 10 10 10 10 10 10 10 10 10 10 CottonHyPep ™ 4601N 5 5 5 5 5 5 5 5 5 5 K₂HPO₄ 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.55.5 5.5 Na₂HPO₄ 3 3 3 3 3 3 3 3 3 3 OD 24 hr 540 nm 2.166 2.154 2.1512.148 2.115 2.120 2.145 2.147 2.140 600 nm 1.940 1.923 1.922 1.922 1.8921.896 1.919 1.922 1.917

As a result, as shown in FIGS. 11a and 11 b, there was no difference inthe growth of the bacterium whether the medium contained NaCl or not.Thus, NaCl was excluded from the final APF medium components.

Example 10 Measurement of Growth Pattern of Clostridium botulinum inFinally Selected APF Medium and Toxin Concentration

Clostridium botulinum was inoculated into the finally selectedClostridium botulinum culture medium (10 g/L glucose, 5 g/L Hy-Pea™7404, 10 g/L UltraPep™ Cotton, 5 g/L HyPep™ 4601N, 5.5 g/L K₂HPO₄, and 3g/L Na₂HPO₄) determined based on the results of Examples 1 to 9, andthen the growth pattern of the bacterium and the toxin concentrationwere measured.

Table 12 shows the time-dependent OD value and toxin concentration ofClostridium botulinum grown in the finally selected APF medium.

TABLE 12 Total Toxin Conc. Time of OD Toxin Conc. in after rupturingCulture (hr) 540 nm 600 nm Supernatant (μg/ml) strain (μg/ml) 0 0 0 0 06 0.0953 0.0393 0.00 0.00 9 0.0648 0.0525 0.00 0.00 12 0.5003 0.44110.00 0.00 14 1.1328 0.9958 2.18 2.04 16 1.6252 1.4484 4.64 10.22 182.3435 2.0215 6.77 18.15 20 2.777 2.4015 8.47 29.26 22 3.3485 2.896 9.4631.86 24 3.5465 3.0695 — 31.73 28 3.452 2.982 — 37.31 36 2.5955 2.24221.20 38.00 48 0.792 0.7224 31.41 38.39

As a result, as shown in Table 12 and FIG. 10, the OD value started toincrease after 12 hours of culture of Clostridium botulinum, and at 24hours of culture, the culture medium showed an OD_(540 nm) of 3.5465 andan OD_(600 nm) of 3.0695. Then, the OD value decreased gradually, and at48 hours of culture, the culture medium showed an OD_(540 nm) of 0.792and an OD_(600 nm) of 0.7224. The toxin concentration in the supernatantof Clostridium botulinum started to increase after 14 hours of cultureand showed a final value of 31.41 μg/ml. When the toxin concentrationwas measured after rupturing the bacterium, the toxin started to beproduced after 5 hours of culture, and the toxin concentration continuedto increase, was maintained at a uniform level after 28 hours ofculture, and showed a final value of 38.39 μg/ml.

In conclusion, the finally selected APF (animal protein-free medium)composition determined based on the results of Examples 1 to 10 issummerized in Table 13.

TABLE 13 Components of Medium g/L Carbon Source Glucose 10 NitrogenHy-Pea ™ 7404 5 Source UltraPep ™ Cotton 10 (Vegetable HyPep ™ 4601N 5Peptone) Mineral K₂HPO₄ 5.5 Na₂HPO₄ 3

Example 11 Growth of Clostridium botulinum in Medium ContainingPlant-Derived Peptone and Porcine Peptone

Although the APF medium composition determined through Examples 1 to 10showed an increased growth rate of the bacterium compared to the mediumthat is in current use, it was taken into account to add a mediumcomponent that further increases the growth rate of the bacterium andhas no risk of causing TSE infection or the like. As a result, a porcinepeptone, which has not been reported to cause TSE infection, was addedto the APF medium, and the growth of the bacterium in the resultingmedium was examined

One or two TSE-free porcine peptones were added to the APF mediumdetermined based on the results of Examples 1 to 10. Specifically,Clostridium botulinum was cultured in the medium composition containingthe plant-derived peptone and porcine peptone Primatone P37 and/or Bactoproteose peptone No. 3 for 24 hours and 48 hours, and the growth of thebacterium was examined by measuring the OD (540 nm, 600 nm) valuesduring the culture (Table 14). In addition, the results of themeasurement were subjected to statistical analysis using statisticalprogram, thereby selecting a medium composition showing the highestgrowth at 24 hours of culture of the bacterium.

Table 14 shows the components of a medium for culture of Clostridiumbotulinum, which contains plant-derived peptones, minerals and porcinepeptones.

TABLE 14 Components Flask No. of Medium g/L 1 2 3 4 5 6 7 8 Glucose 1010 10 10 10 10 10 10 10 Casein 20 — — — — — — — — hydrolysate Yeast 10 —— — — — — — — extract Thioglycollate 5 — — — — — — — — in mediumHy-Pea ™ 5 5 5 5 5 5 5 5 5 7404 UltraPep ™ 10 10 10 10 10 10 10 10 10Cotton HyPep ™ 5 5 5 5 5 5 5 5 5 4601N K₂HPO₄ 5.5 5.5 5.5 5.5 5.5 5.55.5 5.5 5.5 Na₂HPO₄ 3 3 3 3 3 3 3 3 3 Primatone 10 10 10 20 — 20 — — P37Bacto 10 10 10 20 — — 20 10 procine peptone No. 3 OD 24 hr 540 nm 4.9514.625 4.685 4.380 5.469 4.549 4.552 4.426 600 nm 4.436 4.129 4.175 3.9723.011 4.083 4.022 3.934 OD 48 hr 540 nm 1.541 1.595 1.579 2.690 1.2061.811 1.787 1.977 600 nm 1.246 1.317 1.328 2.266 1.042 1.562 1.558 1.697Components Flask No. of Medium g/L 9 10 11 12 13 14 15 Glucose 10 10 1010 10 10 10 10 Casein 20 — — — — — — 20 hydrolysate Yeast 10 — — — — — —10 extract Thioglycollate 5 — — — — — — 5 in medium Hy-Pea ™ 5 5 5 5 5 55 — 7404 UltraPep ™ 10 10 10 10 10 10 10 — Cotton HyPep ™ 5 5 5 5 5 5 5— 4601N K₂HPO₄ 5.5 5.5 5.5 5.5 5.5 5.5 5.5 — Na₂HPO₄ 3 3 3 3 3 3 3 —Primatone 20 10 10 10 10 10 — P37 Bacto 10 — 20 10 10 10 — procinepeptone No. 3 OD 24 hr 540 nm 4.937 4.396 4.934 4.835 4.775 4.699 1.206600 nm 4.396 3.888 4.394 4.319 4.255 4.207 1.011 OD 48 hr 540 nm 2.0721.912 2.312 1.306 1.627 1.546 2.814 600 nm 1.798 1.643 2.025 1.281 1.5451.496 2.373

As a result, as shown in Table 14, when the bacterium was cultured inthe medium further containing porcine peptones, the medium showed an ODvalue higher than that of the medium containing plant-derived peptonesalone. Particularly, the medium showing the highest growth rate of thebacterium was a medium containing 10 g/L Primatone P37 and 10 g/L Bactoproteose peptone, which showed an OD_(540 nm) value of 4.951.

Meanwhile, in order to determine conditions in which the growth of thebacterium can be maximized, the experiment was extended to a responsesurface method, and the results of the response surface method weresubjected to statistical analysis using statistical program. Inaddition, as shown in FIG. 11(A), a response contour plot was drawn, andas a result, conditions in which the growth of the bacterium can bemaximized were found in the experimental range. Furthermore, theconditions in which the growth of the bacterium can be maximized werecalculated. As a result, as shown in FIG. 11(B), the medium containing13.131 g/L of Primatone P37 and 12.727 g/L of Bacto proteose peptone No.3 showed an OD_(540 nm) value of 4.8718, which corresponds to thehighest growth rate of the bacteirum. For convenience of the mediumcomposition, each of the additional medium components was used in anamount of 13 g/L in subsequent experiments.

Example 12 Growth Pattern of Clostridium botulinum in Medium ContainingPlant-Derived Peptone and Porcine Peptone

Using the medium composition containing plant-derived peptones andporcine peptones, determined in Examples 1 to 11, Clostridium botulinumwas cultured, and the growth pattern of the bacterium in the medium wasexamined (see Table 15).

Table 15 shows time-dependent OD values comparing the growth ofClostridium botulinum between the medium that is in current use, themedium containing plant-derived peptone (APF medium) and the finalmedium containing plant-derived peptone and porcine peptone (APF+porcinepeptone).

TABLE 15 medium that is in APF + Porcine Time of current use APF MediumPeptone Culture (hr) OD_(540nm) OD_(600nm) OD_(540nm) OD_(600nm)OD_(540nm) OD_(600nm) 0 0 0 0 0 0 0 9 0.0009 0.0014 0.1101 0.0964 0.25630.2319 12 0.0161 0.0132 0.5537 0.4880 1.3588 1.1892 15 0.1163 0.09751.4508 1.2508 3.5608 3.1432 18 0.3448 0.2911 2.0240 1.7572 5.2240 4.472020 0.5255 0.4468 2.8140 2.4210 6.1450 5.2850 22 1.5292 1.3164 2.95002.5345 6.0950 5.2040 24 2.0528 1.8084 3.1365 2.6970 — — 29 2.8350 2.40902.9415 2.5290 6.9880 6.0920 33 3.0535 2.6030 2.9130 2.5090 3.0902 2.651036 3.1755 2.7115 2.3710 2.0255 2.9205 2.5445 41 3.2015 2.7270 1.48301.2855 2.5010 2.1710 45 3.0450 2.5875 1.3145 1.1305 2.0910 1.8200 602.6696 2.2732 0.6276 0.5800 0.9991 0.9990

As a result, as shown in Table 15 and FIG. 12, at 20 hours of culture ofthe bacterium, the growth of the bacterium in the medium containingplant-derived peptones and porcine peptones was active, and the mediumshowed an OD value which was about 11 times higher than that of themedium that is in current use and about 2.2 times higher than that ofthe medium containing plant-derived peptones alone. At 29 hours ofculture of the bacterium, the peak OD value of the medium containingplant-derived peptones and porcine peptones was at least two timeshigher than that of each of the medium that is in current use and themedium containing plant-derived peptones alone. Thus, it could be seenthat the growth rate of the bacterium in the medium containingplant-derived peptones and porcine peptones was highest among those inthe three kinds of media.

In conclusion, based on the results of Examples 11 and 12, the finallyselected composition comprising porcine peptones in addition to theanimal protein-free (APF) medium is shown in Table 16.

TABLE 16 Composition of Medium Components of Medium g/L Carbon SourceGlucose 10 Nitrogen Source Hy-Pea ™ 7404 5 (Vegetable Peptone)UltraPep ™ Cotton 10 HyPep ™ 4601N 5 Mineral K₂HPO₄ 5.5 Na₂HPO₄ 3Nitrogen Source Primatone P37 13 (Porcine Peptone) Bacto proteosepeptone No. 3 13

INDUSTRIAL APPLICABILITY

As described above, when the medium according to the present invention,which contains plant-derived peptones, porcine peptones and minerals, isused for culture of Clostridium botulinum, the growth rate of thebacterium in the medium is higher than that in each of the medium thatis in current use and the medium containing plant-derived peptone alone.In addition, when the medium of the present invention is used, a highconcentration of botulinum toxin can be produced by culturing thebacterium in a safe manner

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1. A medium composition for culture of Clostridium botulinum, the mediumcomposition comprising: at least one plant-derived peptone selected fromthe group consisting of a garden pea hydrolysate, a cotton seedhydrolysate and a wheat gluten hydrolysate; and a porcine peptone. 2.The medium composition of claim 1, wherein the plant-derived peptone iscomprised with content of 0.1-10 w/v %.
 3. The medium composition ofclaim 1, wherein the medium composition comprising the garden peahydrolysate, the cotton seed hydrolysate and the wheat glutenhydrolysate in the medium composition with a ratio of1:0.24-43.62:0.01-50.57 by weight, provided if the medium compositioncomprising the garden pea hydrolysate, the cotton seed hydrolysate andthe wheat gluten hydrolysate.
 4. The medium composition of claim 1,wherein the porcine peptone in the medium composition is comprised withcontent of 0.2-10 w/v %.
 5. The medium composition of claim 1, whereinthe porcine peptone is a hydrolysate comprising about 54.91-60.69 wt %of peptides having a molecular weight of 500 Da or less, and/or ahydrolysate comprising about 38.48-42.53 wt % of peptides having amolecular weight of 500 Da or less.
 6. The medium composition of claim5, wherein content of the porcine peptone is calculated by weight usingthe following equation 1, provided if the porcine peptone comprises botha hydrolysate comprising about 54.91-60.69 wt % of peptides having amolecular weight of 500 Da or less and a hydrolysate comprising about38.48-42.53 wt % of peptides having a molecular weight of 500 Da orless:B≧−0.625*A+12.5, B≦−1.019*A+53   Equation 1 wherein A: the content(0-5.2 w/v %) of the hydrolysate comprising about 54.91-60.69 wt % ofpeptides having a molecular weight of 500 Da or less; B: the content(0-5.3 w/v %) of the hydrolysate comprising about 38.48-42.53 wt % ofpeptides having a molecular weight of 500 Da or less.
 7. The mediumcomposition of claim 1, wherein the plant-derived peptones or theporcine peptones is subjected to an enzyme treatment.
 8. The mediumcomposition of claim 1, further comprising a carbon source and at leastone mineral selected from the group consisting of K₂HPO₄ (dipotassiumphosphate), Na2HPO₄ (disodium phosphate) and KH₂PO₄ (monopotassiumphosphate).
 9. The medium composition of claim 8, wherein the mineral inthe medium composition is comprised with content of 0.05-3.5 w/v %. 10.A method for producing botulinum toxin, comprising the steps of: (a)culturing Clostridium botulinum using the medium composition of claim 1to produce botulinum toxin; and (b) recovering the produced botulinumtoxin.
 11. The method of claim 10, wherein the culture is performedunder anaerobic conditions.
 12. The method of claim 10, wherein thebotulinum toxin is selected from the group consisting of botulinum toxinserotypes A, B, C, D, E, F and G.